| Objectives:To investigate the effect of Corilagin on the proliferation and migration of vascular smooth muscle cells A7r5 induced by epidermal growth factor(EGF),and to explore the mechanism of Corilagin regulating autophagy through Ras/Raf/ERK1/2signaling pathway to affect cell proliferation and migration,and to explore the cellular and molecular biological mechanism of Corilagin in the prevention and treatment of atherosclerosis.Methods:1.The rat thoracic aortic vascular smooth muscle cells A7r5 were cultured in vitro,and the effects of different concentrations of EGF(5,10,15,20,25,30,35 and40 μg/L)on the proliferation of A7r5 cells at different time points(12,24 and 48h)were detected by CCK-8 method to determine the optimal concentration and time of EGF induction,then the vascular smooth muscle cell proliferation model was replicated.2.The effects of different concentrations of corilagin(12.5,25,50 and 100 μmol/L)on the proliferation of A7r5 cells at different time points(12,24 and 48 h)were detected by CCK-8 method to determine the optimal concentration and time of corilagin administration.3.The effects of different concentrations of corilagin on the proliferation and migration of A7r5 cells induced by EGF were detected by CCK-8 method and cell scratch method.The effects of corilagin on the expression of autophagy-related factors Beclin-1,p62 and LC3 in EGF-induced A7r5 cells were detected by RT-q PCR and Western blot.4.The autophagosome formation of A7r5 cells induced by EGF was observed by transmission electron microscopy.5.The effects of corilagin on the expression of Beclin-1,p62,LC3,Ras,Raf and ERK1/2 in EGF-induced A7r5 cells were detected by RT-q PCR.6.The effect of corilagin on the expression of Beclin-1,p62,LC3,Ras,Raf,p-Raf,ERK1/2 and p-ERK1/2 in A7r5 cells induced by EGF were detected by Western blot.7.After pretreatment with Ras inhibitor FTS,the expression of autophagy-related factors Beclin-1,p62 and LC3 in A7r5 cells was detected by RT-q PCR and Western blot.Results:1.Compared with the normal control group,the proliferation of A7r5 cells was significantly promoted after incubation with different concentrations of EGF for24 h and 48 h,and the proliferation of A7r5 cells was significantly promoted after treatment with 25 μg/L EGF for 24 h.Therefore,25 μg/L EGF was selected to induce the proliferation and migration of A7r5 cells for 24 h.2.The effect of corilagin on the proliferation of A7r5 cells: After 12 h and 24 h of corilagin treatment,compared with the normal control group,there was no significant difference in cell proliferation in the 12.5,25 and 50 μmol/L groups of corilagin,and the 100 μmol/L group of corilagin significantly affected cell viability;after 48 h of corilagin treatment,compared with the normal control group,there was no significant difference in cell proliferation in the 12.5 and 25μmol/L corilagin groups,and the cell proliferation in the 50 and 100 μmol/L corilagin groups was inhibited.Therefore,in the subsequent experiments,corilagin 12.5,25 and 50 μmol/L was selected for 12 h and 24 h as the administration conditions.3.The effect of corilagin on EGF-induced A7r5 cell proliferation: Compared with the normal control group,the proliferation of A7r5 cells in the model group was significantly increased.Compared with the model group,the proliferation of A7r5 cells was weakened after 12 and 24 h of corilagin,and the proliferation of A7r5 cells was also weakened after pretreatment with FTS and 3-MA.4.The effect of corilagin on EGF-induced A7r5 cell migration: Compared with the normal control group,the migration distance of A7r5 cells in the model group was significantly increased.Compared with the model group,the migration distance of A7r5 cells in 12.5,25 and 50 μmol/L corilagin group decreased significantly,and with the increase of corilagin concentration,the migration distance of A7r5 cells decreased gradually,and the migration distance of A7r5 cells decreased after pretreatment with FTS and 3-MA.5.The effect of EGF on the formation of autophagosomes in A7r5 cells: Compared with the normal control group,the nucleus of the model group was irregular,the organelles were significantly reduced,and autophagosomes and autolysosomes were formed.Compared with the model group,50 μmol/L corilagin and 3-MA reduced cell swelling,increased the number of organelles,improved nuclear morphology,and reduced the number of autophagosomes and autolysosomes.6.The effect of corilagin on the expression of autophagy factors in A7r5 cells:Compared with the normal control group,the expression of Beclin-1,LC3 m RNA and protein in the model group increased,and the expression of p62 m RNA and protein decreased;compared with the model group,the expression of Beclin-1and LC3 m RNA and protein decreased,and the expression of p62 m RNA and protein increased in 12.5,25,50 μmol/L corilagin group.7.The effect of corilagin on the expression of Ras/Raf/ERK1/2 signaling pathway related factors in A7r5 cells:(1)RT-q PCR results: Compared with the normal control group,the expression of Ras,Raf and ERK1/2 m RNA in the model group increased;compared with the model group,the expression of Ras,Raf and ERK1/2 m RNA in 12.5 μmol/L,25 μmol/L and 50 μmol/L corilagin groups decreased.(2)Western blot results: Compared with the normal control group,the expression of Ras,p-Raf and P-ERK1/2 protein in the model group increased;compared with the model group,the expression of Ras,p-Raf and P-ERK1/2protein in 12.5 μmol/L,25 μmol/L and 50 μmol/L corilagin groups decreased.There was no significant change in the expression of Raf and ERK1/2 protein.8.After FTS pretreatment,the expression of autophagy factors in EGF-induced damaged A7r5 cells: Compared with the normal control group,the expression of Beclin-1,LC3 m RNA and protein in the model group increased,and the expression of p62 decreased.Compared with the model group,the expression of Beclin-1,LC3 m RNA and protein in 12.5,25,50 μmol/L corilagin group decreased,and the expression of p62 m RNA and protein increased.After FTS pretreatment,the expression of Beclin-1,LC3 m RNA and protein decreased,and the expression of p62 m RNA and protein increased.Conclusions:1.Corilagin can significantly inhibit EGF-induced abnormal proliferation and migration of vascular smooth muscle cells A7r5.2.Corilagin can significantly inhibit EGF-induced autophagy in A7r5 cells,down-regulate the expression of autophagy factors Beclin-1 and LC3,and up-regulate the expression of p62.3.Corilagin can significantly down-regulate the m RNA expression levels of Ras,Raf and ERK1/2 in A7r5 cells induced by EGF,and down-regulate the protein expression levels of Ras,p-Raf and p-ERK1/2.4.The effect of Corilagin on the proliferation and migration of vascular smooth muscle cells induced by EGF is closely related to the inhibition of autophagy by regulating Ras/Raf/ERK1/2 signaling pathway. |
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